Methods of detecting Charcot-Marie Tooth disease type 2A

ABSTRACT

Methods are described for screening a subject for risk of Charcot-Marie-Tooth Disease Type 2A or for diagnosing Charcot-Marie-Tooth disease or a predisposition for developing Charcot-Marie-Tooth disease in a subject, by detecting the presence or absence of a mutation in the mitofusin gene in a biological sample collected from the subject. Methods are also described for detecting the presence of a genetic polymorphism associated with Charcot-Marie-Tooth Disease Type 2A in a sample of patient nucleic acid, by amplifying a mitofusin gene sequence in the patient nucleic acid to produce an amplification product; and identifying the presence of a Charcot-Marie-Tooth Disease Type 2A associated polymorphism in the amplification product.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/731,406, filed Mar. 25, 2010 now U.S. Pat. No. 8,206,922, which is adivisional of U.S. application Ser. No. 10/987,174, filed Nov. 12, 2004,now U.S. Pat. No. 7,727,717, which claims the benefit of U.S.Provisional Application No. 60/520,429, filed on Nov. 14, 2003. Theentire teachings of the above applications are incorporated herein byreference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by grants2P01-NS26630-14 and 2R01-NS29416-09 from the National Institutes ofHealth. The Government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 8, 2013, isnamed 103779-0202_SL.txt and is 28,555 bytes in size.

BACKGROUND OF THE INVENTION

Charcot-Marie-Tooth (CMT) neuropathy, also known as hereditary motor andsensory neuropathy, is a heterogeneous group of inherited diseases ofperipheral nerves. CMT is a common disorder affecting both children andadults. CMT causes significant neuromuscular impairment. It is estimatedthat 1/2500 persons have a form of CMT, making it one of the largestcategories of genetic diseases.

CMT comprises a frequently occurring, genetically heterogeneous group ofperipheral neuropathies, although the clinical picture is ratheruniform. See, Vance et al., The many faces of Charcot-Marie-Toothdisease. Arch Neurol 57, 638-640 (2000). Following electrophysiologicalcriteria, CMT falls into two major forms, the demyelinating CMT type 1with decreased nerve conduction velocities (NCV), and the axonal form,CMT type 2. In contrast to the well known molecular genetic defectscausing the CMT1 phenotype, several genes underlying CMT2 have onlyrecently been identified. So far, seven loci for autosomal dominant CMT2have been assigned to chromosomes 1p35-36 (CMT2A), 3q13-22 (CMT2B),12q23-24 (CMT2C), 7p14 (CMT2D), 8p21 (CMT2E), 7q11-21 (CMT2F), and12q12-13.3 (CMT2G). See, e.g., Ben Othmane et al., Localization of agene (CMT2A) for autosomal dominant Charcot-Marie-Tooth disease type 2to chromosome 1p and evidence of genetic heterogeneity. Genomics 17,370-375 (1993); Kwon et al., Assignment of a second Charcot-Marie-Toothtype II locus to chromosome 3q. Am J Hum Genet 57, 853-858 (1995); Kleinet al., The gene for HMSN2C maps to 12q23-24: a region of neuromusculardisorders. Neurology 60, 1151-1156 (2003); Ionasescu et al., Autosomaldominant Charcot-Marie-Tooth axonal neuropathy mapped on chromosome 7p(CMT2D). Hum Mol Genet 5, 1373-1375 (1996); Mersiyanova et al., A newvariant of Charcot-Marie-Tooth disease type 2 is probably the result ofa mutation in the neurofilament-light gene. Am J Hum Genet 67, 37-46(2000); Ismailov et al., A new locus for autosomal dominantCharcot-Marie-Tooth disease type 2 (CMT2F) maps to chromosome 7q11-q21.Eur J Hum Genet 9, 646-650 (2001).

Currently four genes, involved in CMT2A, CMT2B, CMT2D and CMT2E, havebeen identified. The neurofilament-light gene (NEFL) is responsible forCMT2E, and a large study revealed that NEFL mutations occur in only 2%of CMT patients. See, Jordanova et al., Mutations in the neurofilamentlight chain gene (NEFL) cause early onset severe Charcot-Marie-Toothdisease, Brain 126, 590-597 (2003). Two missense mutations in theRAS-related late-endosomal GTP-binding protein RAB7 have been shown tocause CMT2B in 3 extended families and 2 familial cases with differentethnic backgrounds. See, Verhoeven et al., Mutations in the smallGTP-ase late endosomal protein RAB7 cause Charcot-Marie-Tooth type 2Bneuropathy. Am J Hum Genet 72, 722-727 (2003). Missense mutations in thegene coding for Glycyl tRNA synthetase (GARS) were reported to causeCMT2D and distal hereditary motor neuropathy type VII in differentfamilies. Antonellis et al., Glycyl tRNA Synthetase Mutations inCharcot-Marie-Tooth Disease Type 2D and Distal Spinal Muscular AtrophyType V. Am J Hum Genet 72, 1293-1299 (2003).

In a single Japanese family with a posterior probability supportinglinkage to the CMT2A locus, a missense mutation in the KIF1B-β gene(c.293A>T; Gln98Leu) was found to co-segregate with the disease. Zhao etal., Charcot-Marie-Tooth disease type 2A caused by mutation in amicrotubule motor KIF1Bb. Cell 105, 587-597 (2001). The Leu98 allele wasnot found in 95 healthy control individuals. In addition, the authors ofthis study demonstrated that Kif1B^(+/−) mice developed a chronicperipheral neuropathy resembling the CMT phenotype in humans. Zhao etal. 2001. Yet, no further CMT2A families have been reported with amutation in KIF1B-β. Therefore, it may be desirable to find a differentmethod of diagnosing Charcot-Marie-Tooth disease.

SUMMARY OF THE INVENTION

The present invention includes a method of screening a subject for riskof Charcot-Marie-Tooth Disease Type 2A comprising detecting the presenceor absence of a mutation in the mitofusin gene in a biological samplecollected from the subject; and determining if the subject is at anincreased or decreased risk of Charcot-Marie-Tooth Disease Type 2A dueto the presence of the mutation in the mitofusin gene. The presentinvention also includes methods for detecting the presence of a geneticpolymorphism associated with Charcot-Marie-Tooth Disease Type 2A in asample of patient nucleic acid, comprising amplifying a mitofusin genesequence in the patient nucleic acid to produce an amplificationproduct; and identifying the presence of a Charcot-Marie-Tooth DiseaseType 2A associated polymorphism in the amplification product. Thepresent invention also include methods of diagnosing Charcot-Marie-ToothDisease or a genetic predisposition for developing Charcot-Marie-ToothDisease in a subject, comprising providing a biological sample from thesubject wherein said sample comprises a mitofusin gene; detecting one ormore mutations in the mitofusin gene; and determining that the subjecthas at least one detected mutation in at least one genomic copy of themitofusin gene, wherein the presence of at least one detected mutationin the mitofusin gene is diagnostic for Charcot-Marie-Tooth Disease or agenetic predisposition for developing Charcot-Marie-Tooth Disease in thesubject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B illustrate a transcript map of the CMT2A region onchromosome 1p35.2. FIG. 1A illustrates the physical map with thecontiguous NT_(—)021937 containing KIF1B, typical STR markers, and thescreened genes including MFN2. The CMT2A locus is defined by the markersD1S160 and D1S434. FIG. 1B depicts the genomic structure of MFN2 withsix detected unique mutations within functional domains (white bars);translated mRNA (black bars), untranslated mRNA and alternative splicedexons (grey bars); tel: telomeric; cen: centromeric; TM: transmembranedomain; Cc: coiled-coil.

FIGS. 2A-2E illustrates the pedigrees and detected mutations in fiveCMT2A families, FIGS. 2A-2E disclose SEQ ID NOS 67-76, respectively, inorder of appearance.

FIGS. 3A-3C illustrates the sequence conservation of MFN2 and MFN1 indifferent species related to predicted domains. The sites of theidentified mutations in CMT2A families are indicated by triangles. FIG.3A illustrate three different missense mutations were identified at thebeginning of the GTPase domain. The broken line corresponds to theGTPase starting point. Sequences include those from H. sapiens Mfn2 (SEQID NO:1); M. musculus Mfn2 (SEQ ID NO:2); D. melanogaster (SEQ ID NO:3);C. elegans Mnf2 (SEQ ID NO:4); H. sapiens Mfn1 (SEQ ID NO:5); and M.musculus Mfn1 (SEQ ID NO:6). FIG. 3B depicts two conserved missensemutations in the GTPase domain. Sequences include those from H. sapiensMfn2 (SEQ ID NO:7); M. musculus Mfn2 (SEQ ID NO:8); D. melanogaster (SEQID NO:9); C. elegans Mnf2 (SEQ ID NO:10); H. sapiens Mfn1 (SEQ IDNO:11); and M. musculus Mfn1 (SEQ ID NO:12). FIG. 3C shows a missensemutation occurred at the end of the fzo_mitofusin domain. The blackbackground for this figure indicates highly conserved amino acids. Thescale orientates on the human MFN2 protein sequence (NM_(—)014874).Sequences include those from H. sapiens Mfn2 (SEQ ID NO:13); M. musculusMfn2 (SEQ ID NO:14); D. melanogaster (SEQ ID NO:15); C. elegans Mnf2(SEQ ID NO:16); and H. sapiens Mfn1 (SEQ ID NO:17).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of screening (e.g., diagnosing orprognosing) for diseases, such as Charcot-Marie-Tooth Disease in asubject. The present invention relates to methods for the geneticdiagnosis of Charcot-Marie-Tooth Disease as well as to probes for thegenetic diagnosis of Charcot-Marie-Tooth Disease. Embodiments of thepresent invention are also directed to detecting the presence or absenceof genetic polymorphisms in genes relating to Charcot-Marie-ToothDisease. The present invention relates to data excluding mutations inthe KIF1B gene in six CMT2A families. The lack of KIF1B mutations inthese families illustrate genetic heterogeneity at the CMT2A locus.

One of the embodiments of the present invention includes a method ofscreening a subject for risk of Charcot-Marie-Tooth Disease Type 2Acomprising detecting the presence or absence of a mutation in themitofusin gene in a biological sample collected from the subject.Detecting the presence or absence of a mutation in the mitofusin genecan assist in determining if the subject is at an increased or decreasedrisk of Charcot-Marie-Tooth Disease Type 2A due to the presence of themutation in the mitofusin gene. The detecting step can test forhomozygous or heterozygous mutations. The biological sample can includeboth nucleic and amino acids. The sample can also include a chromosomalnucleic acid. The chromosomal nucleic acid can be Chromosome 1 or afragment thereof. Additional these fragments can include chromosome 1p36and fragments thereof of this fragment. The chromosomal nucleic acid canfurther be defined as being located within the markers D1S160 and D1S434(FIG. 1A). The mutation detected can occur any position in a mitofusingene. These different mutations can include both missense and nonsensemutations and can be located in the gene Mitofusin 2 (MFN2), which islocated 1.65 Mb downstream from the KIF1B locus on chromosome 1p36 (FIG.1). Some of the embodiments of the present invention include mutationsat positions selected from the group consisting of 2219, 839, 751, 493,281, 227 and 205 in a nucleic acid sequence of a mitofusin 2 (geneaccession number AAH17061, incorporated by reference). Those skilled inthe art will appreciate that similar deletions can be made in thehomologous regions of other mitofusin genes, such as mitofusin 1,accession number AAH40557, incorporated by reference. These mutationsfor mitofusin 2 can change the nucleic acid sequence as follows:2219G>C, 839G>A, 751C>G, 493 C>G, 281G>A, 227T>C and 205G>T. Additionalmutations may be located applying the algorithm by Lupas et al.,Predicting coiled coils from protein sequences. Science 252, 1162-1164(1991). Thus, one of skill in the art could determine that a change inthe amino acid sequence could extend the coiled-coil structure thatoccurs at the end of the fzo_mitofusin domain which would indicateCharcot-Marie-Tooth Disease. Additionally, one of skill in the art candetermine a homologous region of a mitofusin gene similar to themutations of the mitofusin 2 gene.

Embodiments of the present invention also include amino acid mutationscaused by mutations in the nucleic acid sequence. These mutations canoccur at positions 740, 280, 251, 165, 76 and 69 in an amino acidsequence of a mitofusin 2 gene, or a homologous region of a mitofusingene. The mutations are based upon the nucleic acid mutations discussedabove. These mutations can result in a missense mutation which causes anamino acid mutation. In particular embodiments, these mutations canresult in the following changes: 740Trp>Ser; 280Arg>His, 251Pro>A1a,165His>Asp, 76Leu>Pro and 69Val>Phe. Another embodiment of the presentinvention includes a method for detecting the presence of a geneticpolymorphism associated with Charcot-Marie-Tooth Disease Type 2A in asample of a patient's nucleic acid. This method can comprise amplifyinga mitofusin gene sequence in the patient nucleic acid to produce anamplification product, and identifying the presence of aCharcot-Marie-Tooth Disease Type 2A associated polymorphism in theamplification product. The polymorphism can be identified by sequencingthe amplification product. Additionally, the amplification product canbe digested with a restriction enzyme so that the Charcot-Marie-ToothDisease Type 2A polymorphism is identified by sequencing a restrictionfragment.

Embodiments of the present invention can also include methods ofdiagnosing Charcot-Marie-Tooth Disease or a genetic predisposition fordeveloping Charcot-Marie-Tooth Disease in a subject. These methods caninclude providing a mitofusin gene from the subject, detecting one ormore mutations in the biological sample, and determining that thesubject has at least one detected mutation in at least genomic copy ofthe mitofusin gene. Thus, a test can be performed to determine if thesubject is homozygous or heterozygous for Charcot-Marie-Tooth Disease.The presence of at least one detected mutation in at least copy of thesequence encoding the mitofusin gene is diagnostic forCharcot-Marie-Tooth Disease or a genetic predisposition for developingCharcot-Marie-Tooth Disease in a subject or the subject's offspring.

Mutations in MFN2 represent the major gene locus for theCharcot-Marie-Tooth neuropathy type 2A. The MFNs, which reside at theouter mitochondrial membrane, have been shown to regulate themitochondrial network architecture by the fusion of mitochondria.Mitochondria represent a tubular and branched membrane network, whichundergoes a dynamically regulated balance between fusion and fissionreactions. MFN2 has one human homologue, MEM1, and highly conservedmembers in different species, including Caenorhabditis elegans and thefuzzy onions (Fzo) gene in Drosophila melanogaster (FIG. 3).

The majority of the identified mutations in CMT2A families were in exons4, 8, and 9 and related to the GTPase domain (FIG. 1B), which has beenshown to be essential for the mitochondrial fusion activity of Mfn2.See, Santel et al., Control of mitochondrial morphology by a humanmitofusin. J Cell Sci 114, 867-874 (2001); Hales et al., Developmentallyregulated mitochondrial fusion mediated by a conserved, novel, predictedGTPase. Cell 90, 121-129 (1997); and Hermann et al., Mitochondrialfusion in yeast requires the transmembrane GTPase Fzo1p. J Cell Biol143, 359-373 (1998). The affected amino acids were conserved in variousspecies (FIG. 3). Analysis of MFN2 by PSORT and MITOPROT revealed amitochondrial targeting signal at the N-terminal site, thus the detectedmutations in CMT2A families V69F, L76P, and R94Q can modulatemitochondrial targeting. One mutation occurred in the fzo_mitofusindomain in exon 19 (FIG. 1B). This mutation can extend the C-terminalcoiled-coil domain, which is required for efficient mitochondrialtargeting.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety.

“Functional polymorphism” as used herein refers to a change in the basepair sequence of a gene that produces a qualitative or quantitativechange in the activity of the protein encoded by that gene (e.g., achange in specificity of activity; a change in level of activity). Thepresence of a functional polymorphism indicates that the subject is atgreater risk of developing a particular disease as compared to thegeneral population. For example, the patient carrying the functionalpolymorphism may be particularly susceptible to chronic exposure toenvironmental toxins that contribute to Charcot-Marie-Tooth Disease. Theterm “functional polymorphism” includes mutations, deletions andinsertions.

The term “Mutation” as used herein sometimes refers to a functionalpolymorphism that occurs in less than five percent of the population,and is strongly correlated to the presence of a gene (i.e., the presenceof such mutation indicating a high risk of the subject being afflictedwith a disease). However, “mutation” is also used herein to refer to aspecific site and type of functional polymorphism, without reference tothe degree of risk that particular mutation poses to an individual for aparticular disease.

Subjects for screening and/or treatment with the present invention are,in general, human subjects, including both female and male subjects. Thesubject may be of any race and any age, including juvenile, adolescent,and adult. It will be appreciated by those skilled in the art that,while the present methods are useful for screening subjects to providean initial indication of the suitability of a patient for a particulartreatment, this information will typically be considered by a clinicianor medical practitioner in light of other factors and experience inreaching a final judgment as to the treatment which any given subjectshould receive.

Suitable subjects include those who have not previously been diagnosedas afflicted with Charcot-Marie-Tooth Disease, those who have previouslybeen determined to be at risk of developing Charcot-Marie-Tooth Disease,and those who have been initially diagnosed as being afflicted withCharcot-Marie-Tooth Disease where confirming information is desired.Thus, it is contemplated that the methods described herein be used inconjunction with other clinical diagnostic information known ordescribed in the art which are used in evaluation of subjects withCharcot-Marie-Tooth Disease or suspected to be at risk for developingsuch disease.

The detecting step may be carried out in accordance with knowntechniques, such as by collecting a biological sample containing DNAfrom the subject, and then determining the presence or absence of DNAencoding or indicative of the mutation in the biological sample. Anybiological sample which contains the DNA of that subject may beemployed, including tissue samples and blood samples, with blood cellsbeing a particularly convenient source.

In general, the step of detecting the polymorphism of interest may becarried out by collecting a biological sample containing DNA from thesubject, and then determining the presence or absence of DNA containingthe polymorphism of interest in the biological sample. Any biologicalsample which contains the DNA of that subject may be employed, includingtissue samples and blood samples, with blood cells being a particularlyconvenient source. The nucleotide sequence of the mitofusin gene isknown and suitable probes, restriction enzyme digestion techniques, orother means of detecting the polymorphism may be implemented based onthis known sequence in accordance with standard techniques. See, e.g.,U.S. Pat. Nos. 6,027,896 and 5,767,248 to A. Roses et al. (Applicantsspecifically intend that the disclosures of all United States patentreferences cited herein be incorporated by reference herein in theirentirety).

Determining the presence or absence of DNA encoding a particularmutation may be carried out with an oligonucleotide probe labeled with asuitable detectable group, and/or by means of an amplification reactionsuch as a polymerase chain reaction or ligase chain reaction (theproduct of which amplification reaction may then be detected with alabeled oligonucleotide probe or a number of other techniques). Further,the detecting step may include the step of detecting whether the subjectis heterozygous or homozygous for the particular mutation. Numerousdifferent oligonucleotide probe assay formats are known which may beemployed to carry out the present invention. See, e.g., U.S. Pat. No.4,302,204 to Wahl et al.; U.S. Pat. No. 4,358,535 to Falkow et al.; U.S.Pat. No. 4,563,419 to Ranki et al.; and U.S. Pat. No. 4,994,373 toStavrianopoulos et al. (applicants specifically intend that thedisclosures of all U.S. Patent references cited herein be incorporatedherein by reference).

Amplification of a selected, or target, nucleic acid sequence may becarried out by any suitable means. See generally, Kwoh et al., Am.Biotechnol. Lab. 8, 14-25 (1990). Examples of suitable amplificationtechniques include, but are not limited to, polymerase chain reaction,ligase chain reaction, strand displacement amplification (see generallyG. Walker et al., Proc. Natl. Acad. Sci. USA 89, 392-396 (1992); G.Walker et al., Nucleic Acids Res. 20, 1691-1696 (1992)),transcription-based amplification (see D. Kwoh et al., Proc. Natl. AcadSci. USA 86, 1173-1177 (1989)), self-sustained sequence replication (or“3SR”) (see J. Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874-1878(1990)), the Qβ replicase system (see P. Lizardi et al., BioTechnology6, 1197-1202 (1988)), nucleic acid sequence-based amplification (or“NASBA”) (see R. Lewis, Genetic Engineering News 12 (9), 1 (1992)), therepair chain reaction (or “RCR”) (see R. Lewis, supra), and boomerangDNA amplification (or “BDA”) (see R. Lewis, supra). Polymerase chainreaction is particularly used.

Polymerase chain reaction (PCR) may be carried out in accordance withknown techniques. See, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202;4,800,159; and 4,965,188. In general, PCR involves, first, treating anucleic acid sample (e.g., in the presence of a heat stable DNApolymerase) with one oligonucleotide primer for each strand of thespecific sequence to be detected under hybridizing conditions so that anextension product of each primer is synthesized which is complementaryto each nucleic acid strand, with the primers sufficiently complementaryto each strand of the specific sequence to hybridize therewith so thatthe extension product synthesized from each primer, when it is separatedfrom its complement, can serve as a template for synthesis of theextension product of the other primer, and then treating the sampleunder denaturing conditions to separate the primer extension productsfrom their templates if the sequence or sequences to be detected arepresent. These steps are cyclically repeated until the desired degree ofamplification is obtained. Detection of the amplified sequence may becarried out by adding to the reaction product an oligonucleotide probecapable of hybridizing to the reaction product (e.g., an oligonucleotideprobe of the present invention), the probe carrying a detectable label,and then detecting the label in accordance with known techniques, or bydirect visualization on a gel. When PCR conditions allow foramplification of all allelic types, the types can be distinguished byhybridization with an allelic specific probe, by restrictionendonuclease digestion, by electrophoresis on denaturing gradient gels,or other techniques.

Ligase chain reaction (LCR) is also carried out in accordance with knowntechniques. See, e.g., R. Weiss, Science 254, 1292 (1991). In general,the reaction is carried out with two pairs of oligonucleotide probes:one pair binds to one strand of the sequence to be detected; the otherpair binds to the other strand of the sequence to be detected. Each pairtogether completely overlaps the strand to which it corresponds. Thereaction is carried out by, first, denaturing (e.g., separating) thestrands of the sequence to be detected, then reacting the strands withthe two pairs of oligonucleotide probes in the presence of a heat stableligase so that each pair of oligonucleotide probes is ligated together,then separating the reaction product, and then cyclically repeating theprocess until the sequence has been amplified to the desired degree.Detection may then be carried out in like manner as described above withrespect to PCR.

DNA amplification techniques such as the foregoing can involve the useof a probe, a pair of probes, or two pairs of probes which specificallybind to DNA containing the functional polymorphism, but do not bind toDNA that does not contain the functional polymorphism. Alternatively,the probe or pair of probes could bind to DNA that both does and doesnot contain the functional polymorphism, but produce or amplify aproduct (e.g., an elongation product) in which a detectable differencemay be ascertained (e.g., a shorter product, where the functionalpolymorphism is a deletion mutation). Such probes can be generated inaccordance with standard techniques from the known sequences of DNA inor associated with a gene linked to Charcot-Marie-Tooth Disease or fromsequences which can be generated from such genes in accordance withstandard techniques.

It will be appreciated that the detecting steps described herein may becarried out directly or indirectly. Other means of indirectlydetermining allelic type include measuring polymorphic markers that arelinked to the particular functional polymorphism, as has beendemonstrated for the VNTR (variable number tandem repeats).

Kits for determining if a subject is or was (in the case of deceasedsubjects) afflicted with or is or was at increased risk of developingCharcot-Marie-Tooth Disease will include at least one reagent specificfor detecting for the presence or absence of at least one functionalpolymorphism as described herein and instructions for observing that thesubject is or was afflicted with or is or was at increased risk ofdeveloping Charcot-Marie-Tooth Disease if at least one of the functionalpolymorphisms is detected. The kit may optionally include one or morenucleic acid probes for the amplification and/or detection of thefunctional polymorphism by any of the techniques described above, withPCR being currently utilized.

Molecular biology comprises a wide variety of techniques for theanalysis of nucleic acid and protein sequences. Many of these techniquesand procedures form the basis of clinical diagnostic assays and tests.These techniques include nucleic acid hybridization analysis,restriction enzyme analysis, genetic sequence analysis, and theseparation and purification of nucleic acids and proteins (See, e.g., J.Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: ALaboratory Manual, 2 Ed., Cold spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989).

Most of these techniques involve carrying out numerous operations (e.g.,pipetting, centrifugation, and electrophoresis) on a large number ofsamples. They are often complex and time consuming, and generallyrequire a high degree of accuracy. Many a technique is limited in itsapplication by a lack of sensitivity, specificity, or reproducibility.

For example, the complete process for carrying out a DNA hybridizationanalysis for a genetic or infectious disease is very involved. Broadlyspeaking, the complete process may be divided into a number of steps andsub-steps. In the case of genetic disease diagnosis, the first stepinvolves obtaining the sample (e.g., saliva, blood or tissue). Dependingon the type of sample, various pre-treatments would be carried out. Thesecond step involves disrupting or lysing the cells which releases thecrude DNA material along with other cellular constituents.

Generally, several sub-steps are necessary to remove cell debris and tofurther purify the DNA from the crude sample. At this point severaloptions exist for further processing and analysis. One option involvesdenaturing the DNA and carrying out a direct hybridization analysis inone of many formats (dot blot, microbead, microplate, etc.). A secondoption, called Southern blot hybridization, involves cleaving the DNAwith restriction enzymes, separating the DNA fragments on anelectrophoretic gel, blotting the DNA to a membrane filter, and thenhybridizing the blot with specific DNA probe sequences. This procedureeffectively reduces the complexity of the genomic DNA sample, andthereby helps to improve the hybridization specificity and sensitivity.Unfortunately, this procedure is long and arduous. A third option is tocarry out an amplification procedure such as the polymerase chainreaction (PCR) or the strand displacement amplification (SDA) method.These procedures amplify (increase) the number of target DNA sequencesrelative to non-target sequences. Amplification of target DNA helps toovercome problems related to complexity and sensitivity in genomic DNAanalysis. After these sample preparation and DNA processing steps, theactual hybridization reaction is performed. Finally, detection and dataanalysis convert the hybridization event into an analytical result.

Nucleic acid hybridization analysis generally involves the detection ofa very small number of specific target nucleic acids (DNA or RNA) withan excess of probe DNA, among a relatively large amount of complexnon-target nucleic acids. A reduction in the complexity of the nucleicacid in a sample is helpful to the detection of low copy numbers (i.e.10,000 to 100,000) of nucleic acid targets. DNA complexity reduction isachieved to some degree by amplification of target nucleic acidsequences. (See, M. A. Innis et al., PCR Protocols: A Guide to Methodsand Applications, Academic Press, 1990, Spargo et al., 1996, Molecular &Cellular Probes, in regard to SDA amplification). This is becauseamplification of target nucleic acids results in an enormous number oftarget nucleic acid sequences relative to non-target sequences therebyimproving the subsequent target hybridization step.

The actual hybridization reaction represents one of the most importantand central steps in the whole process. The hybridization step involvesplacing the prepared DNA sample in contact with a specific reporterprobe at set optimal conditions for hybridization to occur between thetarget DNA sequence and probe.

Hybridization may be performed in any one of a number of formats. Forexample, multiple sample nucleic acid hybridization analysis has beenconducted in a variety of filter and solid support formats (See Beltz etal., Methods in Enzymology, Vol. 100, Part et al., Eds., Academic Press,New York, Chapter 19, pp. 266-308, 1985). One format, the so-called “dotblot” hybridization, involves the non-covalent attachment of target DNAsto a filter followed by the subsequent hybridization to a radioisotopelabeled probe(s). “Dot blot” hybridization gained wide-spread use overthe past two decades during which time many versions were developed (seeAnderson and Young, in Nucleic Acid Hybridization—A Practical Approach,Hames and Higgins, Eds., IRL Press, Washington, D.C. Chapter 4, pp.73-111, 1985). For example, the dot blot method has been developed formultiple analyses of genomic mutations (EPA 0228075 to Nanibhushan etal.) and for the detection of overlapping clones and the construction ofgenomic maps (U.S. Pat. No. 5,219,726 to Evans).

Additional techniques for carrying out multiple sample nucleic acidhybridization analysis include micro-formatted multiplex or matrixdevices (e.g., DNA chips) (see M. Barinaga, 253 Science, pp. 1489, 1991;W. Bains, 10 Bio/Technology, pp. 757-758, 1992). These methods usuallyattach specific DNA sequences to very small specific areas of a solidsupport, such as micro-wells of a DNA chip. These hybridization formatsare micro-scale versions of the conventional “dot blot” and “sandwich”hybridization systems.

The micro-formatted hybridization can be used to carry out “sequencingby hybridization” (SBH) (see M. Barinaga, 253 Science, pp. 1489, 1991;W. Bains, 10 Bio/Technology, pp. 757-758, 1992). SBH makes use of allpossible n-nucleotide oligomers (n-mers) to identify n-mers in anunknown DNA sample, which are subsequently aligned by algorithm analysisto produce the DNA sequence (See, Drmanac U.S. Pat. No. 5,202,231).

There are two formats for carrying out SBH. The first format involvescreating an array of all possible n-mers on a support, which is thenhybridized with the target sequence. The second format involvesattaching the target sequence to a support, which is sequentially probedwith all possible n-mers. Both formats have the fundamental problems ofdirect probe hybridizations and additional difficulties related tomultiplex hybridizations.

Southern, (United Kingdom Patent Application GB 8810400, 1988; E. M.Southern et al., 13 Genomics 1008, 1992), proposed using the firstformat to analyze or sequence DNA. Southern identified a known singlepoint mutation using PCR amplified genomic DNA. Southern also describeda method for synthesizing an array of oligonucleotides on a solidsupport for SBH. However, Southern did not address how to achieveoptimal stringency conditions for each oligonucleotide on an array.

Drmanac et al., (260 Science 1649-1652, 1993), used the second format tosequence several short (116 bp) DNA sequences. Target DNAs were attachedto membrane supports (“dot blot” format). Each filter was sequentiallyhybridized with 272 labeled 10-mer and 11-mer oligonucleotides. Wideranges of stringency conditions were used to achieve specifichybridization for each n-mer probe. Washing times varied from 5 minutesto overnight using temperatures from 0° C. to 16° C. Most probesrequired 3 hours of washing at 16° C. The filters had to be exposed from2 to 18 hours in order to detect hybridization signals. The overallfalse positive hybridization rate was 5% in spite of the simple targetsequences, the reduced set of oligomer probes, and the use of the moststringent conditions available.

Currently, a variety of methods are available for detection and analysisof the hybridization events. Depending on the reporter group(fluorophore, enzyme, radioisotope, etc.) used to label the DNA probe,detection and analysis are carried out fluorimetrically,colorimetrically, or by autoradiography. By observing and measuringemitted radiation, such as fluorescent radiation or particle emission,information may be obtained about the hybridization events. Even whendetection methods have very high intrinsic sensitivity, detection ofhybridization events is difficult because of the background presence ofnon-specifically bound materials. Thus, detection of hybridizationevents is dependent upon how specific and sensitive hybridization can bemade. Concerning genetic analysis, several methods have been developedthat have attempted to increase specificity and sensitivity.

One form of genetic analysis is analysis centered on elucidation ofsingle nucleic acid polymorphisms or (“SNPs”). Factors favoring theusage of SNPs are their high abundance in the human genome (especiallycompared to short tandem repeats, (STRs)), their frequent locationwithin coding or regulatory regions of genes (which can affect proteinstructure or expression levels), and their stability when passed fromone generation to the next (Landegren et al., Genome Research, Vol. 8,pp. 769-776, 1998).

A SNP is defined as any position in the genome that exists in twovariants and the most common variant occurs less than 99% of the time.In order to use SNPs as widespread genetic markers, it is crucial to beable to genotype them easily, quickly, accurately, and cost-effectively.It is of great interest to type both large sets of SNPs in order toinvestigate complex disorders where many loci factor into one disease(Risch and Merikangas, Science, Vol. 273, pp. 1516-1517, 1996), as wellas small subsets of SNPs previously demonstrated to be associated withknown afflictions.

Numerous techniques are currently available for typing SNPs (for review,see Landegren et al., Genome Research, Vol. 8, pp. 769-776, (1998), allof which require target amplification. They include direct sequencing(Carothers et al., BioTechniques, Vol. 7, pp. 494-499, 1989),single-strand conformation polymorphism (Orita et al., Proc. Natl. Acad.Sci. USA, Vol. 86, pp. 2766-2770, 1989), allele-specific amplification(Newton et al., Nucleic Acids Research, Vol. 17, pp. 2503-2516, (1989),restriction digestion (Day and Humphries, Analytical Biochemistry, Vol.222, pp. 389-395, 1994), and hybridization assays. In their most basicform, hybridization assays function by discriminating shortoligonucleotide reporters against matched and mismatched targets. Manyadaptations to the basic protocol have been developed. These includeligation chain reaction (Wu and Wallace, Gene, Vol. 76, pp. 245-254,1989) and minisequencing (Syvanen et al., Genomics, Vol. 8, pp. 684-692,1990). Other enhancements include the use of the 5′-nuclease activity ofTaq DNA polymerase (Holland et al., Proc. Natl. Acad. Sci. USA, Vol. 88,pp. 7276-7280, 1991), molecular beacons (Tyagi and Kramer, NatureBiotechnology, Vol. 14, pp. 303-308, 1996), heat denaturation curves(Howell et al., Nature Biotechnology, Vol. 17, pp. 87-88, 1999) and DNA“chips” (Wang et al., Science, Vol. 280, pp. 1077-1082, 1998).

An additional phenomenon that can be used to distinguish SNPs is thenucleic acid interaction energies or base-stacking energies derived fromthe hybridization of multiple target specific probes to a single target.(see R. Ornstein et al., “An Optimized Potential Function for theCalculation of Nucleic Acid Interaction Energies”, Biopolymers, Vol. 17,2341-2360 (1978); J. Norberg and L. Nilsson, Biophysical Journal, Vol.74, pp. 394-402, (1998); and J. Pieters et al., Nucleic Acids Research,Vol. 17, no. 12, pp. 4551-4565 (1989)). This base-stacking phenomenon isused in a unique format in the current invention to provide highlysensitive Tm differentials allowing the direct detection of SNPs in anucleic acid sample.

Additional methods have been used to distinguish nucleic acid sequencesin related organisms or to sequence DNA. For example, U.S. Pat. No.5,030,557 by Hogan et al. disclosed that the secondary and tertiarystructure of a single stranded target nucleic acid may be affected bybinding “helper” oligonucleotides in addition to “probe”oligonucleotides causing a higher Tm to be exhibited between the probeand target nucleic acid. That application however was limited in itsapproach to using hybridization energies only for altering the secondaryand tertiary structure of self-annealing RNA strands which if leftunaltered would tend to prevent the probe from hybridizing to thetarget.

With regard to DNA sequencing, K. Khrapko et al., Federation of EuropeanBiochemical Societies Letters, Vol. 256, no. 1,2, pp. 118-122 (1989),for example, disclosed that continuous stacking hybridization resultedin duplex stabilization. Additionally, J. Kieleczawa et al., Science,Vol. 258, pp. 1787-1791 (1992), disclosed the use of contiguous stringsof hexamers to prime DNA synthesis wherein the contiguous stringsappeared to stabilize priming. Likewise, L. Kotler et al., Proc. Natl.Acad. Sci. USA, Vol. 90, pp. 4241-4245, (1993) disclosed sequencespecificity in the priming of DNA sequencing reactions by use of hexamerand pentamer oligonucleotide modules. Further, S. Parinov et al.,Nucleic Acids Research, Vol. 24, no. 15, pp. 2998-3004, (1996),disclosed the use of base-stacking oligomers for DNA sequencing inassociation with passive DNA sequencing microchips. Moreover, G. Yershovet al., Proc. Natl. Acad. Sci. USA, Vol. 93, pp. 4913-4918 (1996),disclosed the application of base-stacking energies in SBH on a passivemicrochip. In Yershov's example, 10-mer DNA probes were anchored to thesurface of the microchip and hybridized to target sequences inconjunction with additional short probes, the combination of whichappeared to stabilize binding of the probes. In that format, shortsegments of nucleic acid sequence could be elucidated for DNAsequencing. Yershov further noted that in their system the destabilizingeffect of mismatches was increased using shorter probes (e.g., 5-mers).Use of such short probes in DNA sequencing provided the ability todiscern the presence of mismatches along the sequence being probedrather than just a single mismatch at one specified location of theprobe/target hybridization complex. Use of longer probes (e.g., 8-mer,10-mer, and 13-mer oligos) were less functional for such purposes.

An additional example of methodologies that have used base-stacking inthe analysis of nucleic acids includes U.S. Pat. No. 5,770,365 by Laneet al., wherein is disclosed a method of capturing nucleic acid targetsusing a unimolecular capture probe having a single stranded loop and adouble stranded region which acts in conjunction with a binding targetto stabilize duplex formation by stacking energies.

Despite the knowledge of base-stacking phenomenon, applications asdescribed above have not resulted in commercially acceptable methods orprotocols for either DNA sequencing or the detection of SNPs forclinical purposes. We provide herein such a commercially useful methodfor making such distinctions in numerous genetic and medicalapplications by combining the use of base-stacking principles andelectronically addressable microchip formats.

Kits useful for carrying out the methods of the present invention will,in general, comprise one or more oligonucleotide probes and otherreagents for carrying out the methods as described above, such asrestriction enzymes, optionally packaged with suitable instructions forcarrying out the methods.

The present invention also provides a method of conducting a clinicaltrial on a plurality of human subjects or patients. Such methodsadvantageously permit the refinement of the patient population so thatadvantages of particular treatment regimens (typically administration ofpharmaceutically active organic compound active agents) can be moreaccurately detected, particularly with respect to particularsub-populations of patients. In general, such methods compriseadministering a test active agent or therapy to a plurality of subjects(a control or placebo therapy typically being administered to a separatebut similarly characterized plurality of subjects) and detecting thepresence or absence of at least one mutation or polymorphism asdescribed above in the plurality of subjects. The polymorphisms may bedetected before, after, or concurrently with the step of administeringthe test therapy. The influence of one or more detected polymorphisms orabsent polymorphisms on the test therapy can then be determined on anysuitable parameter or potential treatment outcome or consequence,including but not limited to: the efficacy of said therapy, lack of sideeffects of the therapy, etc.

In describing the mutations disclosed herein in the novel proteinsdescribed herein, and the nucleotides encoding the same, the namingmethod is as follows: [nucleic acid replaced] [nucleic acid number insequence of known sequence] [alternate nucleic acid]. For example, forthe 2219 position is guanine and is replaced with an cytosine.

The present invention is explained in greater detail in the followingnon-limiting examples.

EXAMPLE 1 Identification of Mutations in the Mitofusion 2 GeneAssociated with CMT 2A

In all the families identified, different missense mutations in the geneMitofusin 2 (MFN2) were located. The gene Mitofusin 2 (MFN2) is located1.65 Mb downstream from the KIF1B locus on chromosome 1p36 (FIG. 1).

Methods

Patients

The CMT2A families DUK662, DUK1706, DUK1241, CMT156 were studied. TheRussian family RU45 was ascertained at the Research Center for MedicalGenetics, Moscow. The Turkish family CMT166 was identified incollaboration of the University of Istanbul and the University ofAntwerp. Controls consisted of unrelated spouses of CMT families andunrelated individuals of Turkish nationality with no clinical signs ofperipheral neuropathies. All samples were collected with informedconsent. Tissue for RT-PCR was obtained from a human tissue bank at theDepartment of Neuropathology, University Hospital, Rhineland-WestphalianTechnical University. The study was approved by each collaboratorsinstitutional review board or equivalent.

Mutation Screening

All PCR primers were designed with the web-based primer3 algorithm. PCRreactions followed standard protocols. PCR products were visualized on1.5% agarose gels stained with ethidium bromide. The reaction productswere purified applying the Qiaquick PCR purification kit (Quiagen,Hilden, Germany). Amplified DNA samples were directly sequenced applyingthe Big dye Terminator reaction kit (Applied Biosystems, Foster City,USA) on an ABI 3730.

Genes were sequenced for mutation screening in coding exons and flankingintronic sequences in both directions (forward and reverse).

RT-PCR

For transcript analysis at the cDNA level, total RNA was isolated fromblood samples using the PAXgene Blood RNA Kit (PreAnalytiX,Hombrechtikon, Switzerland) and RNeasy (Qiagen, Hilden, Germany). ThemRNA was reverse transcribed to cDNA with random primers (ReverseTranscription System, Promega, Madison, USA). The KIF1B-β and MFN2 cDNAswere amplified with primer sets, which produced overlapping products.

Genotyping and Linkage Analysis

For genotyping of family RU45 the following microsatellite markers wereused to test linkage to the CMT2A locus: D1S2663 (AFMa210xg9), D1S508(AFMal28ye9), D1S2667 (AFMa224wg9), D1S228(AFM196xb4). A newly designedSTR marker at contig AC019262 was amplified by the primers AC019262-F:GGAGTGCATTTCTGCTTGGTAG (SEQ ID NO: 19) and AC019262-R:AACACTTGGCTTATACCTTTTCTAG (SEQ ID NO:20). All PCR reactions wereperformed following standard procedures. Two-point linkage analysis wasperformed by the programs MLINK and ILINK (LINKAGE package, version5.1). LOD scores were calculated under the assumption of equal markerallele frequencies, and the disease was assessed as an autosomaldominant trait with a 0.0001 disease allele frequency. The FASTLINKpackage (version 4.1P) was used for multipoint analysis of data.

Electronic Database Information

Accession numbers and URLs for data presented herein are as follows:

BLAST searches, www.ncbi.nlm.nih.gov/BLAST

Ensembl Genome Browser, www.ensembl.org

Entrez Protein, www.ncbi.nlm.nih.gov/entrez (mitofusin 2, Homo sapiens[accession number AAH17061]; mitofusin 2, Mus musculus [accession numberAAM88577], mitochondrial assembly regulatory factor, Drosophilamelanogaster [accession number AAM00196]; mitofusin 2, Caenorhabditiselegans [accession number NP_(—)495161]; mitofusin 1, Homo sapiens[accession number AAH40557]; mitofusin 1, Mus musculus [accession numberNP-077162] 1229555.1

ExPASy Molecular Biology Server, www.expasy.ch

GenBank, www.ncbi.nlm.nih.gov/Genbank ([accession number NT_(—)015074],UBE4B [accession number NM_(—)006048], PEX [accession numberNM_(—)004565], TARDBP [accession number NM_(—)007375], PMSLC [accessionnumber NM_(—)002685], FRAP1 [accession number NM_(—)004958], KIAA1337[accession number XM_(—)052561], FBXO2 [accession number NM_(—)012168],FBG3 [accession number NM_(—)033182], FBXO6 [accession numberNM_(—)001286], CLCN6 [accession number NM_(—)001286], NPPA [accessionnumbers NM_(—)006172], NPPB [accession number NM_(—)002521], TNFRSF8[accession number NM_(—)001243], KIAA0453 [accession numberXM_(—)044546], KIF1B [accession number NM_(—)015074], MFN2 [accessionnumber NM_(—)014874], and MFN1 [accession number NM_(—)033540])

Genome Data Base, www.gdb.org

HUGE database, www.kazusa.or.jp/huge (for KIAA1337, KIAA0453)

Inherited Peripheral Neuropathies Mutation Database,

molgen-www.uia.ac.be/CMTMutations

MITOPROT, ihg.gsf.de/ihg/mitoprot.html (for prediction of mitochondrialtargeting sequences in MFN2)

NCBI Aceview, www.ncbi.nih.gov/IEB/Research/Acembly

NCBI dbEST database, www.ncbi.nlm.nih.gov/dbEST/index.html

NCBI dbSNP database, www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=snp

NCBI for protein access,

NCBI RefSeq project, www.ncbi.nih.gov/RefSeq/

Online Mendelian Inheritance in Man (OMIM)

Pfam, pfam.wustl.edu/index.html (for fzo_mitofusin domain [accessionnumber PF04799.2], GTP binding domain [accession number PF00009]), andP-loop motif)

Primer3, www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi

PSORT, psort.nibb.ac.jp/form.html (for prediction of mitochondrialtargeting sequences in MFN2)

SMART, smart.embl-heidelberg.de

SWISS-PROT, www.expasy.org/sprot/

TMHMM, www.cbs.dtu.dk/services/TMHMM/, (for prediction of transmembranehelices in MFN2)

Unigene, www.ncbi.nlm.nih.gov/UniGene

UCSC genome browser, genome.ucsc.edu

Family Data and Haplotype Analysis

The original CMT2A family (DUK662), three previously reported familiesfrom Italy (CMT156) and Northern America (DUK1241, DUK1706), and twonewly ascertained pedigrees originating from Russia (RU45) and Turkey(CMT166) were studied (FIGS. 2, 3). Linkage analysis to the CMT2A locusfor all six families provided LOD scores ranging from 2.20 to 5.88(Table 1).

TABLE 1 Primers and RT-PCR primers for MFN2 SEQ SEQ Name Forward primerID Reverse primer ID Mit-1 CCATGATGCAGTGGGAGTC 21 GCTTGACTGCATCCCAGAC 22Mit-2 GCAACTCCCCAATACCTCAA 23 GAGACTTGCCACCAGAGGTC 24 Mit-3TGATTCTCCCCAAAGCATTC 25 TATGACTTCCCTGGGAGCAG 26 Mit-4bATCTTCTACCAGCCGCCATT 27 GGATTGAAAATGGGTCACCA 28 Mit-4CCTTCCAGACTTGGGACTGT 29 GCCTGGAACGTTCTGTGAC 30 Mit-5ACTGGCAACATTGCACTGAA 31 GTCTCCCATTCACCTCCACA 32 Mit-6CCACTGTGCTGTGATGCAG 33 AGGGACCCTGGCCTAGATTA 34 Mit-7/8GTCCCAGGTCTGTTCTCAGC 35 CACTAGATCCAGGGGTGCAG 36 Mit-9TCCCAAAGTGCTGGGATTAC 37 TCTCAGCATCCCCTTCTCAG 38 Mit-10CTGAGAAGGGGATGCTGAGA 39 TCACTGCAGACTGGGAGATG 40 Mit-11TCTGTGCCTCCCCAGCTC 41 GGTGGAGCACCCTTGTCTC 42 Mit-12 ATTTCTGGCATCCCCTCTTG43 TGAAAGGCTGAGAAGTCCCTA 44 Mit-13 GCCATCTGCTAGGATCTCTCC 45TGTCTCTGTGGCTTCTACTGTCA 46 Mit-14 CCCAGCAGTGACAGTAGAAGC 47CCAGAACCTGAAGGTATCGAGT 48 Mit-15 TGGTAGAGCCCTGTCTCCAA 49TTAGGGAACCCCCGTTTTAG 50 Mit-16 GAGCCACTCTGTGTCCCTGT 51CAGTGGACTGTGGAGTGTGG 52 Mit-17 GAAACATGAAGGCTCCTTGG 53AGAGAGATGGGGAAGGGAGA 54 Mit-18 AGGAGATTCTGCCAAACCAG 55TTTGTGTCCACACCCAAGAC 56 Mit-19 GGTGTGTGTCAAGCGTCCTT 57GATGAAGCACTGAGCCAACA 58 MitRT ex2-6 CATGATGCCTACCCTGTGAA 59CCAGACAAAACTTGTCAATCCA 60 MitRT ex6-12 TGTGATGTGGCCCAACTCTA 61GACACCTGCCTCTCCACTTC 62 MitRT ex12-16 CGGGAGCAGCAGGTTTACT 63CATGGAGGTCCTGGATGTCA 64 MitRT ex16-19 TTGATGGGCTACAATGACCA 65TGCTTCATTCTCTTGGCAGT 18

The flanking markers for the CMT2A locus were originally designated bythe family DUK662 and were later refined to a 10.0 cM region byrecombinants in family CMT156 (ref. 3, 16). The Turkish family CMT166provided a further reduction of the CMT2A locus to 9.3 cM, defined bythe markers D1S160 and D1S434 (FIG. 1A). Summarized clinical andelectrophysiological data are shown in Table 2.

TABLE 2 Missense mutations (A) and intragenic SNPs (B) detected in MFN2Exon/ In- CMT2A Nucleotide Amino acid Ethnic dbSNP tron Family changechange origin number A 4 CMT166 c.205G > T Val69Phe Turkish 4 DUK1706c.227T > C Leu76Pro Northern American 4 RU45 c.281G > A Arg94Gln Russian8 CMT156 c.751C > G Pro251Ala Italian 9 DUK1241 c.839G > A Arg280HisNorthern American 19 DUK662 c.2219G > C Trp740Ser Northern American

Description of a Newly Ascertained CMT2A Family

Pedigree RU45 represents a CMT2A family originating from Russia. In allpatients of the family the disease is characterized by limb weakness andsevere atrophy of the peroneal, distal femoral, and distal hand muscles.Further “stocking and glove” sensory loss, absence of ankle andcarpo-radial reflexes, pes cavus, and steppage gait were observed. Oneaffected (marked in grey in FIG. 3) suffered from cerebral palsy; thushis CMT status was not established clearly. Electrophysiologicalanalysis of three affected females demonstrated normal NCV values forthe motor median nerve and moderately decreased for the tibial nerve(Table 2). The maximum two-point LOD score of 3.55 was obtained for themarker AC019262, lying near D1S434.

Mutation Screening in KIF1B

Direct sequencing of the amplified coding exons of KIF1B-β in thefamilies DUK662, DUK1706, DUK1241, RU45, CMT156, and CMT166 revealed nomutations. In addition, direct sequencing of the KIF1B-β cDNA of twoaffected subjects in families CMT156 and CMT166 revealed no additionalsequence variations, deletions or insertions. RT-PCR with primersspanning the entire gene and producing overlapping PCR products did notdisclose evidence for additional exons in the vicinity of KIF1B-β inhuman samples of peripheral nerve tissue. However, this experimentdemonstrated a formerly described splice variant of KIF1B-β lacking exon25. This shorter splice variant of KIF1B-β was present in cDNA retrievedfrom blood, peripheral nerve, spinal cord, brain, and muscle tissue. Thelonger isoform was expressed in muscle, spinal cord, and brain.

Several single nucleotide polymorphisms (SNP) distributed over theentire gene were detected in coding exons and flanking intronicsequences in patients and 40 healthy controls. As the KIF1B geneconsists of a head and two alternatively spliced tails, α and β,mutations in KIF1B-β were also excluded by sequencing.

Mutation Detection in Mitofusin 2 (MFN2)

The refined chromosomal region of 9.6 cM contains at least 55 known orpredicted genes. Candidate genes with known expression in the nervoussystem were prioritized for mutation analysis. The following genes werescreened for mutations in affected individuals from the examinedfamilies: UBE4B, PEX, TARDBP, PMSLC, FRAP1, KIAA1337, FBXO2, FBG3,FBXO6, CLCN6, NPPA, NPPB, TNFRSF8, KIAA0453, and MFN2 (FIG. 1). In thegene MFN2, six different missense mutations were identified in the sixfamilies. In family DUK662 a c.2219G>C substitution (Trp740Ser)completely co-segregated with the CMT2 phenotype, but was not evident in250 healthy Caucasian controls. Applying the algorithm by Lupas et al.,the exchange from the aromatic tryptophan to the small polar serine waspredicted to extend the coiled-coil structure that occurs at the end ofthe fzo_mitofusin domain (FIG. 1). The mutations in families DUK1241(c.839G>A, Arg280His) and CMT156 (c.751C>G, Pro251Ala) were found in theGTPase domain of the protein. Both Pro251 and Arg280 amino acids arehighly conserved in Drosophila melanogaster and Caenorhabditis elegans,suggesting functional importance (FIG. 3). In family RU45, an Arg94Glnmutation was caused by a transition of G>A at position 281 (c.281G>A).This amino acid marks the predicted beginning of the GTPase domain andis conserved in the GTPase domain of MFN1, a homolog protein of MFN2(FIG. 3). The mutation in family DUK1706 (Leu76Pro, c.227T>C) also liesat the beginning of the GTPase domain. The Leu76 allele is alsoconserved in mammals and D. melanogaster (FIG. 3). In the Turkishfamily, CMT166, an exchange of G>T substitutes Valine for Phenylalanine(c.205G>T, Va169Phe). The Va169 allele is similarly highly conserved inMFN2 (FIG. 3). No mutations were detected in at least 250 healthycontrol samples.

Expression of MFN2 in Human Neural Tissue

By RT-PCR the presence of MFN2 transcripts was shown in human muscle,sural nerve, spinal cord, and brain. A formerly predicted alternativeexon 4b (FIG. 1 b) was verified in all samples. This alternativetranscript begins translation at exon 4b, leading to a shortening (96amino acids) at the N-terminal of the protein.

EXAMPLE 2 Additional Mutations Found in CMT 2A Individuals

Using the methods described herein, additional mutations were identifiedin MFN2 in CMT2 patients. One mutation was a 493 C>G change, resultingin 165His>Asp. This mutation is associated with CMT2 and mild spasticfeatures in the clinical examination, strongly implying the involvementof the central nervous system. The mutation segregated in a largeAustralian family and was not found in 500 control chromosomes.

Additional mutations are described in Supplementary Tables 1 and 2below.

SUPPLEMENTARY TABLE 1 Observed intragenic SNPs in KIF1B-β. Effect oncoding Exon/Intron Nucleotide change sequence dbSNP number  4 c.183 −2delTT 5′-splice site —  4 c.285C > G Ala95Ala — IVS5 c.429 + 26G > A —rs4846209 IVS5 c.430 − 31A > T — — IVS7 c.720 + 17C > T — — IVS13c.1296 + 38A > G — rs3748576 IVS18 c.1723 + 125A > G — — IVS36 c.3813 −53A > T — rs4846215 38 c.4161A > G Pro1387Pro — 46 c.5163C > AThr1721Thr —

SUPPLEMENTARY TABLE 2 MFN2 mutations found in 36 additional CMT2families that were too small for linkage analysis. Family DUK1265DUK2007 DUK2128 DUK2158 DUK2173 DUK2176 DUK2451 Ethnic origin NorthAmerica North America North America North America North AmericaIran/Iraq North America Mutation in MFN2 c.2219G > C; c.2219G > C;c.839G > A; c.1252C > T; c.280C > T; c.821G > A; c.314C > T; Trp740SerTRP740Ser Arg280His Arg418Stop Arg94Trp Arg274Gln Thr105Met Exon 19 19 9 12  4  9 5 Age at onset (years) <10   7-47 28  1 <10  13  3-15 Distalweakness and +/++ +/++ +/+ ++/+++ ++/+++ −/++ +/+++ atrophy, UL/LLDistal sensory loss + + + + + + + Proximal muscle normal normal normalnormal normal normal normal strength Other symptoms — — — visualmigraine — ataxia, scoliosis impairment Achilles tendon absent absentabsent absent absent absent absent reflex Motor NCV, not obtained 40-4949 52 47 58 47-52 Median nerve (m/s) +, mild; ++, moderate, severe; UL,upper limbs; LL, lower limbs; NCV, nerve conduction velocity

The Arg418X change in MFN2, described above in Supplementary Table 2,caused a premature termination of translation in on of the CMT2patients. The clinical phenotype of this patient included early age atonset, vocal cord paresis with hoarse voice, and visual impairment. Thevisual impairment is due to pathologic changes of the retina thatresembles phenotypes known from mitochondrial disease and also fromoptic atrophy. Therefore, a portion of patients diagnosed as Leberhereditary optic atrophy without mutations in the mitochondrial genomemight well have mutations in MFN2.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

What is claimed is:
 1. A method of identifying a human subject forincreased risk of Charcot-Marie-Tooth Disease Type 2A, comprising:amplifying nucleic acid from a mitofusin 2 gene in a biological samplefrom the subject with at least one oligonucleotide that specificallybinds to a mitofusin 2 gene comprising a mutation but does not bind to amitofusin 2 gene that does not comprise the mutation; detecting thepresence of the mutation in the mitofusin 2 gene, wherein the mutationresults in a change in the amino acid sequence of SEQ ID NO: 66 selectedfrom the group consisting of Trp740Ser, His165His, Leu76Pro, andVa169Phe; and identifying the subject as having an increased risk ofCharcot-Marie-Tooth Disease Type 2A based on the presence of themutation in the mitofusin 2 gene.
 2. The method according to claim 1,wherein the mutation in the mitofusin 2 gene is selected from the groupconsisting of G2219C, C493G, T227C, and G205T.
 3. The method of claim 1,wherein the detecting further comprises detecting whether the subject ishomozygous or heterozygous for the mutation.
 4. A method of diagnosingCharcot-Marie-Tooth Disease or a genetic predisposition for developingCharcot-Marie-Tooth Disease in a human subject, comprising: amplifyingnucleic acid from a mitofusin 2 gene in a biological sample from thesubject with at least one oligonucleotide that specifically binds to amitofusin 2 gene comprising a mutation but does not bind to a mitofusin2 gene that does not comprise the mutation; detecting the presence ofthe mutation in the mitofusin 2 gene, wherein the mutation results in achange in the amino acid sequence of SEQ ID NO: 66 selected from thegroup consisting of Trp740Ser, His165His, Leu76Pro, and Va169Phe; anddiagnosing the subject as having Charcot-Marie-Tooth Disease or agenetic predisposition for developing Charcot-Marie-Tooth Disease basedon the presence of the mutation in the mitofusin 2 gene.
 5. The methodaccording to claim 4, further comprising detecting whether said subjectis heterozygous or homozygous for the mutation.
 6. The method accordingto claim 4, wherein the mutation in the mitofusin 2 gene is selectedfrom the group consisting of G2219C, C493G, T227C and G205T.